Koduri R S, Tien M
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802, USA.
J Biol Chem. 1995 Sep 22;270(38):22254-8. doi: 10.1074/jbc.270.38.22254.
We have investigated the lignin peroxidase-catalyzed oxidation of guaiacol and the role of veratryl alcohol in this reaction by steady-state and pre-steady-state methods. Pre-steady-state kinetic analyses demonstrated that guaiacol is a good substrate for both compounds I and II, the two- and one-electron oxidized enzyme intermediates, respectively, of lignin peroxidase. The rate constant for the reaction with compound I is 1.2 x 10(6) M-1s-1. The reaction of guaiacol with compound II exhibits a Kd of 64 microM and a first-order rate constant of 17 s-1. Oxidation of guaiacol leads to tetraguaiacol formation. This reaction exhibits classical Michaelis-Menten kinetics with a Km of 160 microM and a kcat of 7.7 s-1. Veratryl alcohol, a secondary metabolite of ligninolytic fungi, is capable of mediating the oxidation of guaiacol. This was shown by steady-state inhibition studies. Guaiacol completely inhibited the oxidation of veratryl alcohol, whereas veratryl alcohol had no corresponding inhibitory effect on guaiacol oxidation. In fact, at low guaiacol concentrations, veratryl alcohol stimulated the rate of guaiacol oxidation. These results collectively demonstrate that veratryl alcohol can serve as a mediator for phenolic substrates in the lignin peroxidase reaction.
我们通过稳态和预稳态方法研究了木质素过氧化物酶催化的愈创木酚氧化反应以及藜芦醇在该反应中的作用。预稳态动力学分析表明，愈创木酚是木质素过氧化物酶的两种氧化酶中间体（分别为双电子和单电子氧化的中间体）即化合物I和化合物II的良好底物。与化合物I反应的速率常数为1.2×10⁶ M⁻¹s⁻¹。愈创木酚与化合物II的反应表现出64 μM的解离常数（Kd）和17 s⁻¹的一级速率常数。愈创木酚的氧化导致四愈创木酚的形成。该反应呈现出典型的米氏动力学，米氏常数（Km）为160 μM，催化常数（kcat）为7.7 s⁻¹。藜芦醇是木质素分解真菌的次生代谢产物，能够介导愈创木酚的氧化。稳态抑制研究表明了这一点。愈创木酚完全抑制了藜芦醇的氧化，而藜芦醇对愈创木酚的氧化没有相应的抑制作用。事实上，在低浓度愈创木酚条件下，藜芦醇促进了愈创木酚的氧化速率。这些结果共同表明，藜芦醇可以作为木质素过氧化物酶反应中酚类底物的介质。
